We propose to develop a novel automated platform to enable rapid, sensitive, user-friendly, and cost-effective screening for the antibiotic resistant bacteria that frequently cause serious healthcare associated infections. Every year, about 100,000 deaths result from 2 million serious healthcare associated infections at an estimated cost of $35B. About 20,000 fatalities are caused by the bacterium methicillin-resistant Staphylococcus aureus (MRSA). Many healthy individuals are colonized with MRSA. When these MRSA carriers are hospitalized, the resistant microbe can cause serious infections for the carrier and others in the hospital. Several recent studies have shown that hospitals can significantly decrease the number of healthcare associated infections by using rapid tests to screen admitted patients for MRSA colonization and then treating the colonized patient with rigorous infection control procedures. New rapid MRSA tests based on PCR are fast, but are either too expensive or too complex to be implemented in most hospitals. Traditional microbiological methods for screening patients for MRSA are inexpensive but are too slow (1-2 days) to most effectively lower infection rates. We recently completed a Phase I project demonstrating the feasibility of a novel, rapid, simple, and cost-effective method for MRSA screening that should for the first time allow any hospital to implement rapid testing for infection control. The platform technology uses low-cost non-magnified digital imaging to rapidly count individual fluorescently labeled S. aureus cells. The test determines whether a patient's sample contains MRSA by testing for S. aureus cells that can grow in antibiotic-containing media during a brief incubation. The rapid (3.3 hr) Phase I MRSA test's analytical sensitivity is comparable to PCR tests (10 CFU/sample). When applied to clinical nasal samples (n=163), the test showed high clinical sensitivity (95%) and specificity (98%) comparable to the on-market PCR tests. We also designed, developed, and integrated a fully functional automated bench top analyzer prototype and developed and manufactured a low-cost consumable prototype for the MultiPath MRSA test. Here we propose to build on our Phase I work to develop a prototype automated platform and MRSA test cartridge that can be used in subsequent Phase III beta field trials. Specifically we aim to (1) further decrease time to results and develop optimized stable reagents; (2) develop a low-cost MultiPath MRSA/SA test cartridge; (3) develop a low-cost automated beta MultiPath analyzer; (4) integrate and verify the platform functionality; and (5) demonstrate the performance of the MultiPath MRSA/S. aureus (MRSA/SA) test. Achieving these Specific Aims will enable subsequent Phase III beta field testing that will in turn support a future regulatory submission. The MultiPath platform will also be able to accommodate a broad menu of tests for agents that cause healthcare associated infections (e.g., vancomycin resistant Enterococcus and C. difficile), infectious viruses, food pathogens, toxins, molecular disease markers, and drug discovery targets.